Proteomic study on protective mechanism of ischemic preconditioning to ischemia-reperfusion lung injury

Objective To investigate the change of protein expression of lung tissue of rabbit after ischemic preconditioning (IP) and try to elucidate the potential protective mechanism of IP. Methods 12 domestic rabbits were randomly divided into group IP and group control (6 rabbits in each group). All the left lungs were afflicted by ische mia-reperfusion injury except that those in group IP were subject to IP prior to ischemic phase. 2-DE was employed to separate the total protein of the lung tissue. PDQuest analysis software was used to distinguish the differently expressed protein spot. MALDI-TOF-MS and Mascot database searching were exploited to identify these proteins. Results 1) IP attenuated the ischemia-reperfusion lung injury. 2) The proteomic analysis showed 35 target proteins, of which 17 were characterized such as phosphatidylinositol 3-kinase(PI3k) delta catalytic subunit. Conclusions 1) Proteomic is a promising tool to investigate the IP and ischemia-reperfusion lung injury. 2) That IP inhibits inflammatory cascades through phosphatidylinositol 3-kinase signal transduction pathway may be one of its protective mechanism. Foundation item: Project(2004036433) supported by the Postdoctoral Science Foundation of China; project(B2004024) supported by the Science Foundation of Public Health Bureau of Hunan Province     

In Vitro Antimetastatic Effect of Phosphatidylinositol 3-Kinase Inhibitor ZSTK474 on Prostate Cancer PC3 Cells

Tumor metastasis is the main cause of lethality of prostate cancer, because conventional therapies like surgery and hormone treatment rarely work at this stage. Tumor cell migration, invasion and adhesion are necessary processes for metastasis. By providing nutrition and an escape route from the primary site, angiogenesis is also required for tumor metastasis. Phosphatidylinositol 3-kinases (PI3Ks) are well known to play important roles in tumorigenesis as well as metastasis. ZSTK474 is a specific PI3K inhibitor developed for solid tumor therapy. In the present report, antimetastatic activities of ZSTK474 were investigated in vitro by determining the effects on the main metastatic processes. ZSTK474 exhibited inhibitory effects on migration, invasion and adhesive ability of prostate cancer PC3 cells. Furthermore, ZSTK474 inhibited phosphorylation of Akt substrate-Girdin, and the secretion of matrix metalloproteinase (MMP), both of which were reported to be closely involved in migration and invasion. On the other hand, ZSTK474 inhibited the expression of HIF-1α and the secretion of vascular endothelial growth factor (VEGF), suggesting its potential antiangiogenic activity on PC3 cells. Moreover, we demonstrated the antiangiogenesis by determining the effect of ZSTK474-reduced VEGF on tube formation of human umbilical vein endothelial cells (HUVECs). In conclusion, ZSTK474 was demonstrated to have potential in vitro antimetastatic effects on PC3 cells via dual mechanisms: inhibition of metastatic processes including cell migration, invasion and adhesion, and antiangiogenesis via blockade of VEGF secretion.     

Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C

The nucleotide sequence of a 4.3 kb fragment downstream of the LIG4 gene of Candida albicans has been determined. This fragment contains two entire ORFs (ORF1 and ORF2) and a truncated one (ORF3). ORF1 (1029 bp; EMBL databank, Accession No. AJ277539) encodes a putative protein of 343 amino acids with a high degree of similarity to phosphatidylinositol-specific phospholipases C (PI-PLC) of bacterial origin and, to a lesser degree, to similar proteins from trypanosome, fly and human. Isolated ORF1 confers PI-PLC activity to Escherichia coli transformants. ORF2 (1572 bp; EMBL databank, Accession No. AJ277538) predicts a protein of 524 amino acids with high similarity along most of the entire length to Ydr393w from Saccharomyces cerevisiae. This protein carries a domain with significant similarity to several cytoskeleton proteins of different origins. YDR393w (SHE9) is an orphan gene whose overexpression compromises cell growth. ORF3 appears to encode the homologue of the well-conserved proteasomal 26S regulatory subunit.     

Dual Inhibition of MEK and PI3K Pathway in KRAS and BRAF Mutated Colorectal Cancers

Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. Genetic mutations in the phosphatidylinositol-3 kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways are frequently implicated in CRC. Targeting the downstream substrate MEK in these mutated tumors stands out as a potential target in CRC. Several selective inhibitors of MEK have entered clinical trial evaluation; however, clinical activity with single MEK inhibitors has been rarely observed and acquired resistance seems to be inevitable. Amplification of the driving oncogene KRAS(13D), which increases signaling through the ERK1/2 pathway, upregulation of the noncanonical wingless/calcium signaling pathway (Wnt), and coexisting PIK3CA mutations have all been implicated with resistance against MEK inhibitor therapy in KRAS mutated CRC. The Wnt pathway and amplification of the oncogene have also been associated with resistance to MEK inhibitors in CRCs harboring BRAF mutations. Thus, dual targeted inhibition of MEK and PI3K pathway effectors (mTOR, PI3K, AKT, IGF-1R or PI3K/mTOR inhibitors) presents a potential strategy to overcome resistance to MEK inhibitor therapy. Many clinical trials are underway to evaluate multiple combinations of these pathway inhibitors in solid tumors.     

Evidence for Altered Phosphoinositide Signaling-Associated Molecules in the Postmortem Prefrontal Cortex of Patients with Schizophrenia

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3β (GSK3β) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3β expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.     

大豆肌醇磷脂制备优化条件的研究

利用乙醇-正己烷双溶剂体系对大豆磷脂中肌醇磷脂进行分离。对分离条件进行了优化研究,在乙醇浓度为55%、无水乙酸钠的量为0.05g,正己烷用量为20mL,乙醇用量为30mL,10.00g大豆混合磷脂中肌醇磷脂的含量可以从26.00%提高到73.89%。并对双溶剂体系的分离度进行测定,在此条件下,分离度可达18.1。     

A simple and cost-effective gram-scale chromatographic method for the purification of soybean phospholipids

A method was developed for the preparative fractionation of soybean lecithin to enable the study of the functional properties of pure soybean phospholipids. Hereby, a coarse and irregularly shaped silica gel was used as the stationary phase, whereas the mobile phase consisted of three mixtures of hexane, 2-propanol, and water with increasing polarity. These solvents were included in a step gradient, which was formed by an isocratic pump connected to a solvent switcher. With this system, two grams of soybean lecithin were fractionated. The purity was evaluated by analytical high-performance liquid chromatography, and the recovery was estimated from concentration determinations by flow injection analysis. From these results, it was concluded that 60 to 75% of the three major soybean phospholipids could be recovered with a purity of at least 93%. Only 1.5 L of solvents were needed for both the column equilibration and the elution of all soybean phospholipids. Senior research associate of the Belgian National Fund for Scientific Research (N.F.W.O.).